Corydalis tomentella Franch. Exerts anti-inflammatory and analgesic effects by regulating the calcium signaling pathway

Ethnopharmacological relevance Corydalis tomentella Franch. is a perennial cespitose plant commonly used to treat stomachaches as a folk medicine. The C . tomentella total alkaloids have good protective effects against acute liver injury and potential anti-hepatoma and anti-Alzheimer's disease activities. Aim of the study To establish an effective purification process for total alkaloids from C. tomentella and investigate the mechanism of their anti-inflammatory effects. Materials and methods Corydalis tomentella were purified using macroporous resin. Then the crude and purified C. tomentella extracts (cCTE and pCTE) were qualitatively analyzed using UPLC-Triple-TOF-MS/MS. The cCTE and pCTE were used to investigate and compare their anti-inflammatory effects on lipopolysaccharide (LPS)-induced RAW264.7?cells. Doses at 100, 200 and 400?mg/kg/d of pCTE were used to study their anti-inflammatory and analgesic activities in mice with xylene-induced ear swelling and acetic acid-induced writhing tests. Content of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were determined both in RAW264.7?cells and mice. Network pharmacology was used to predict the anti-inflammatory mechanism of C. tomentella , and the key enzymes were validated using qPCR and Western Blot analysis . Concentration of intracellular Ca 2+ was detected using flow cytometric analysis. Results The C. tomentella total alkaloid purity increased from 6.29% to 47.34% under optimal purification conditions. A total of 54 alkaloids were identified from CTE. Both cCTE and pCTE could suppress the LPS-induced production of NO, IL-6, IL-1β, and TNF-α in RAW264.7?cells. The pCTE exhibited a more potent anti-inflammatory effect; it also inhibited pain induced by xylene and acetic acid in mice. The calcium signaling pathway is associated with the anti-inflammatory and analgesic activities of C. tomentella . The mRNA expression of nitric oxide synthase (NOS) 2, NOS3 and calmodulin1 (CALM1) was regulated by C. tomentella through the reduction of inflammation-induced Ca 2+ influx, and it also exhibited a more pronounced effect than the positive control (L-N G -nitro arginine methyl ester). Conclusions Purified C. tomentella extract shows anti-inflammatory effect both in vitro and in vivo. It exerts anti-inflammatory and analgesic effects through the calcium signaling pathway by down-regulating NOS2 and CALM1 expression and up-regulating NOS3 expression in LPS-induced RAW264.7?cells, and decreasing intracellular Ca 2+ concentration.

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